Expression of a heterologous DNA sequence in a plant host is dependent upon the presence of an operably-linked promoter that is functional within the plant host. Choice of the promoter sequence will determine when and where within the plant the heterologous DNA sequence is expressed. Where continuous expression is desired throughout the cells of a plant, a constitutive promoter is utilized. Where gene expression in response to a stimulus is desired, an inducible promoter is the regulatory element of choice. Where expression in a specific tissue or organ is desired, a tissue-preferred promoter is used. Such a tissue-preferred promoter may be inducible. Expression during a particular developmental stage may be achieved with a developmentally-regulated promoter. Additional regulatory sequences upstream and/or downstream from the core promoter sequence can be included in expression cassettes of transformation vectors to bring about varying levels of expression of heterologous nucleotide sequences in a transgenic plant. See, for example, U.S. Pat. No. 5,850,018. Regulatory sequences may also be useful in controlling temporally- and/or spatially-differentiated expression of endogenous DNA.
In grain crops of agronomic importance, seed formation is the ultimate goal of plant development. Seeds are harvested for use in food, feed, and industrial products. The utility and value of those seeds are determined by the quantity and quality of protein, oil, and starch contained therein. In turn, the quality and quantity of seed produced may be affected by environmental conditions at any point prior to fertilization through seed maturation. In particular, stress at or around the time of fertilization may have substantial impact on seed development.
Stresses to plants may be caused by both biotic and abiotic agents. For example, biotic causes of stress include infection with a pathogen, insect feeding, parasitism by another plant such as mistletoe, and grazing by ruminant animals. Abiotic stresses include, for example, excessive or insufficient available water, insufficient light, temperature extremes, synthetic chemicals such as herbicides, excessive wind, extremes of soil pH, limited nutrient availability, and air pollution. Yet plants survive and often flourish, even under unfavorable conditions, using a variety of internal and external mechanisms for avoiding or tolerating stress. Plants' physiological responses to stress reflect changes in gene expression.
While manipulation of stress-induced genes may play an important role in improving plant tolerance to stresses, it has been shown that constitutive expression of stress-inducible genes has a severe negative impact on plant growth and development when the stress is not present. (Kasuga et al., (1999) Nature Biotechnology 17(3):287-291) Therefore, there is a need in the art for promoters driving expression which is temporally- and/or spatially-differentiated, to provide a means to control and direct gene expression in specific cells or tissues at critical times, especially to provide stress tolerance or avoidance.
In particular, drought and/or density stress of maize often results in reduced yield, typically from plant failure to set and fill seed in the apical portion of the ear, a condition known as “tip kernel abortion” or colloquially as “nosing back.” To stabilize plant development and grain yield under unfavorable environments, manipulation of hormones and carbon supply to the developing ear and its kernels is of interest. Thus there is a need for promoters which drive gene expression in female reproductive tissues under abiotic stress conditions.
Improvement of crop plants with multiple transgenes is of increasing interest. This is sometimes known as gene “stacking” and provides opportunities for the manipulation of plant physiology to meet a variety of challenges during the lifecycle of the transformed plant. For example, a single maize hybrid may comprise recombinant DNA constructs conferring not only insect resistance, in the transformed plant's ability to produce an insecticidal toxin derived from Bacillus thuringiensis, but also resistance to a specific herbicide, through incorporation of a Streptomyces hygroscopicus gene that detoxifies glufosinate. Importantly, appropriate regulatory sequences are needed to drive the desired expression of each of these or other transgenes of interest. Furthermore, it is important that regulatory elements be distinct from each other. Concerns associated with the utilization of similar regulatory sequences to drive expression of multiple genes include, but are not restricted to: (a) pairing along homologous regions, crossing-over and loss of the intervening region either within a plasmid prior to integration, or within the plant genome, post-integration; (b) hairpin loops caused by two copies of the sequence in opposite orientation adjacent to each other, again with possibilities of excision and loss of these regulatory regions; (c) competition among different copies of the same promoter region for binding of promoter-specific transcription factors or other regulatory DNA-binding proteins.
Thus, there is a continuing need for promoters which will drive gene expression in the appropriate tissues, at the proper time, to the desired degree, and in response to the relevant stimuli.